Identification of Microproteins in Hep3B Cells at Different Cell Cycle Stages

Microproteins are generated from
small open reading frames and
turn out to play various vital biological functions. As an essential
biological event of eukaryotic cells, the cell cycle is involved in
cell replication and division. For such a highly regulated event,
microproteins associated with cell cycle regulation remained unclarified.
Utilizing a combination of bottom-up and top-down proteomics, we analyzed
microproteins at specific cell cycle stages of Hep3B cells. A total
of 657 microproteins were identified under three cell cycle stages,
including 151 in the G0/G1 stage, 163 in the S stage, and 132 in the
G2/M stage. The annotation of these microproteins showed their cell
cycle-specific functions, such as translation, nuclear assembly, chromatin
organization, and the G2/M transition of the mitotic cell cycle. Meanwhile,
more than 50% of identified microproteins were ncRNA-encoded. These
nonannotated novel microproteins contain several function domains,
such as the nucleoside diphosphate kinase domain, the high mobility
group domain, and the DNA-binding domain. This suggested the potential
functions of these novel microproteins in specific cell cycle stages.
This study presented a large-scale profile of microproteins at different
cell cycle stages from Hep3B and may provide new perspectives on the
regulation mechanism of the cell cycle. Liquid chromatography–mass
spectrometry data were deposited to ProteomeXchange using the identifier
PXD030286.

微蛋白（microproteins）由小型开放阅读框（small open reading frames）编码生成，并被证实具备多种关键生物学功能。作为真核细胞的核心生物学事件，细胞周期参与细胞的复制与分裂过程。针对这一受精密调控的生命过程，目前与细胞周期调控相关的微蛋白仍未得到充分阐释。
本研究结合自下而上与自上而下蛋白质组学（proteomics）技术，对Hep3B细胞的特定细胞周期阶段的微蛋白进行了分析。最终在三个细胞周期阶段中共鉴定出657种微蛋白，其中G0/G1期151种、S期163种、G2/M期132种。对这些微蛋白的功能注释结果显示，它们具备细胞周期特异性功能，例如翻译过程、核组装、染色质组织以及有丝分裂细胞周期的G2/M期转换。同时，超过50%的鉴定得到的微蛋白由非编码RNA（non-coding RNA, ncRNA）编码。这些未被注释的新型微蛋白携带有多种功能结构域，例如核苷二磷酸激酶结构域、高迁移率族结构域以及DNA结合结构域，这提示上述新型微蛋白在特定细胞周期阶段中具备潜在生物学功能。
本研究构建了Hep3B细胞不同细胞周期阶段的大规模微蛋白表达谱，可为细胞周期调控机制的研究提供全新视角。本研究的液相色谱-质谱联用（liquid chromatography–mass spectrometry）数据已通过标识符PXD030286提交至ProteomeXchange数据库。